Project Summary: The current Electron Paramagnetic Resonance imaging system developed as a prototype in vivo oxygen imaging scanner with anatomic correlates obtained from conventional Magnetic Resonance Imaging (MRI) and additional information such as blood vessel density makes use of a combined resonator for both the modalities allowing reliable coregistration of spatial information from both imaging scans. Subsequent developments focused on improving the spatial and temporal resolutions. The EPR imaging system with the developments during the last year now has the following capabilities: A) An image data set can be obtained in 2 minutes permitting the collection of about 8-10 images after a single bolus injection of the tracer Oxo63, allowing the assessment of temporal changes in tumor oxygen status. B) monitoring changes in tumor oxygen status longitudinally over a period of several days-weeks to assess drug induced changes in oxygen status and correlate with treatment efficacy. With these improvements, w have utilized the system for longitudinal monitoring of tumor oxygen status, blood vessel density and the corresponding changes in response to treatment with chemotherapeutic drugs which impact tumor oxygen status and blood vessel density. Based on these capabilities, the following three experiments are being pursued: 1) Assessment of cycling (acute) hypoxia in tumors: Tumors, as a result of their aberrant vasculature have both chronic (diffusion limited) and cycling (acute) hypoxia. While chronic hypoxia is diffusion limited, acute hypoxia has been shown to coner phenotypes which display resistance to treatment and also a more malignant phenotype. While this phenomenon has either been studied using invasive approaches such as insertion of oxygen sensing electrodes or such a pattern enforced experimentally, imaging techniques so far have not been able to study and distinguish the two types of hypoxia. In a study designed to examine this phenomenon using EPR, two tumor models (SCC VII and HT 29) implanted in mice were studied to assess the levels of both chronic and cycling hypoxia. MRI studies were sequentially carried out to obtain anatomic correlates as well as blood vessel density. Histological correlates from tumor sections were also evaluated for blood vessel density and vascular integrity. Air-Carbogen-Air challenge via respiration was also imposed to examine the spatio-temporal responses to tumor oxygen to distinguish chronic vs cycling hypoxic regions. The studies reveal that both tumor types studied exhibited cycling hypoxia, withtyhe SCC VII transplant exhibiting fluctuations to a large magnitude compared to HT 29 tumors. The relatively stronger pericyte coverage in HT 29 tumor vasculature was accounted for the observed differences. 2) Effect of the anti-angiogenic drug, sunitinib on tumor oxygenation and blood vessel density: Ant-angiogenic drugs have been shown to transiently increase tumor oxygen levels after treatment initiation through a process called vascular re-normalization. This is followed by a steady decrease in oxygen resulting from loss of blood vessels. The vascular renormalization period is a window in time for synergy with radiotherapy or chemotherapy. We have evaluated the effect of sunitinib, an anti-angiogenic drug in C3H mice implanted with squamous cell carcinoma (SCC) on the hind leg and monitored changes in oxygen and blood vessel density on several days after treatment initiation and compared with control untreated tumor bearing mice. These studies showed: a) it is possible to longitudinally monitor changes in tumor oxygen and blood vessel density;b) blood vessel density decreases after treatment with sunitinib;and d) there is a transient increase in tumor oxygenation after sunitinib treatment. These data are in agreement with the immunohistochemical data obtained on the markers for blood vessel density. An important finding is that EPR imaging studies can identify the vascular renormalization period in tumors during which time we find that there is a significant synergy when radiotherapy is administered. Another finding is that fluctuations in oxygen are minimized during the period of vascular re-normalization. 3) Effect of the mTOR inhibitor, rapamycin on tumor oxygenation and blood vessel density: Rapamycin is in active investigation in many tumor types in humans. Some of its effects on tumors include autophagy and tumor vessel damage. EPR imaging and MRI studies were conducted on SCC tumor bearing mice during treatment with rapamycin. The studies show that rapamycin has significant influence in tumor vessel damage as early as one day after treatment initiation. However, the tumor vessel decrease was associated with a transient but significant increase in tumor oxygenation in agreement with results which suggest that there is a normalization of tumor vasculature which increases tumor oxygenation.